WO2021140615A1 - Novel recombinant bacteriophage - Google Patents

Novel recombinant bacteriophage Download PDF

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WO2021140615A1
WO2021140615A1 PCT/JP2020/000469 JP2020000469W WO2021140615A1 WO 2021140615 A1 WO2021140615 A1 WO 2021140615A1 JP 2020000469 W JP2020000469 W JP 2020000469W WO 2021140615 A1 WO2021140615 A1 WO 2021140615A1
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phage
gene
recombinant bacteriophage
protein
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哲朗 近藤
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哲朗 近藤
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    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

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  • Phage that infects E. coli is used in the phage display method, and functions because foreign proteins and peptides fused with the coat protein of the phage can be presented on the surface of the phage in a form that can interact with other molecules. It has become one of the most powerful methods for exploring motifs. However, since a complicated method such as ELISA is used in the phage detection step in the functional motif screening process, there is a drawback that the screening requires enormous labor and time.
  • Non-Patent Documents 1 and 2 T7 phages (Non-Patent Documents 1 and 2), T4 phages (Non-Patent Documents 3), or lambda phage (Non-Patent Documents 4) have been visualized using fluorescent proteins such as GFP. There is a report to detect it. However, since the number of fluorescent proteins expressed on the phage is extremely small, 1 to 3, the fluorescence intensity is weak, and the number of fluorescent proteins per phage cannot be controlled, so that it is not constant and is accurate using the fluorescence intensity as an index. Cannot be screened or detected.
  • Non-Patent Documents 1 to 3 have a fluorescent protein fused to a capsid, there is a drawback that the fluorescent protein causes steric damage to the presented protein when used in the phage display method. There is. Similarly, a recombinant bacteriophage expressing the luciferase enzyme gene described in Patent Document 1 on the surface of a capsid may cause such steric hindrance.
  • the present inventor inserts a gene for a fluorescent protein, which is a labeling protein, or a gene for an enzyme that catalyzes a coloring reaction into a specific gene region of a T7 phage so as to be expressible.
  • a fluorescent protein which is a labeling protein
  • a gene for an enzyme that catalyzes a coloring reaction into a specific gene region of a T7 phage so as to be expressible.
  • the fluorescent protein is green fluorescent protein (GFP), and the enzyme that catalyzes the coloring reaction is one of luciferase, ⁇ -glucuronidase (GUS), ⁇ -galactosidase, and horseradish peroxidase.
  • GFP green fluorescent protein
  • the present invention also relates to a method using the random peptide phage library of recombinant bacteriophage T7 described in (8) below or the random peptide phage library of (9) and (10) below.
  • a random peptide is placed in the gp10 gene region of the genome of recombinant bacteriophage T7 in which the gene of the labeled protein is expressively inserted into the gp17 gene region of the genome of bacteriophage T7.
  • the number of fluorescent proteins expressed per phage molecule is as small as 1 to 3, but in the recombinant phage of the present invention, a large number of fluorescent proteins expressed by one order higher than in the conventional method are expressed, so that fluorescence is achieved. There is a difference in strength at each stage. Further, in the conventional method, the number of fluorescent proteins per phage molecule cannot be controlled, the number of fluorescent proteins differs depending on the phage, and the fluorescence intensity is not constant. Since the same number of fluorescent proteins are always expressed in the phage, screening with an accurate constant fluorescence intensity can be realized.
  • the unbound fluorescent protein is added to each round in which the selected phage is replicated and proliferated during the panning process in the screening. There is no need to separate the phages that emit the desired fluorescence each time, and it is not necessary to verify that the fluorescent protein is definitely bound to all the selectively amplified phages in each round.
  • the fluorescent protein is expressed in a structural portion different from the capsid moiety expressing the random peptide in the library portion, which causes a steric disorder of binding to the target. It has an excellent effect that it does not overlook the binding to the target, which is an important element in the phage display method.
  • FIG. 3 shows a solid fluorescence micrograph of a plaque of Escherichia coli infected with recombinant T7gp17-sfGFP.
  • Bacteriophage T7 is a lytic phage having double-stranded linear genomic DNA, binds to LPS on the surface of Escherichia coli, and injects DNA into Escherichia coli to infect.
  • the head of the phage that stores the genomic DNA is formed by the association of 415 G10 proteins, and the tail is added below it.
  • bacteriophage or "phage” is a virus that has evolved to use bacteria in nature as a means of replicating them.
  • the phage attaches the phage itself to the bacterium, injects its DNA into the bacterium, and induces the bacterium to replicate hundreds or even thousands of times the phage. Do. This is also called phage amplification.
  • genes of enzymes that catalyze luminescence or color reaction can be used, for example, luciferase (Luc), ⁇ -glucuronidase (GUS), ⁇ -galactosidase (Gal), horseradish peroxidase (HRP), alkaline phosphatase ( Examples include commercially available genes such as AP).
  • DNA encoding a peptide having a binding property to a fluorescent / photoprotein or a fluorescent dye can be used.
  • a biotinylated tag sequence peptide acceptor peptide of Escherichia coli biotin-ligase BirA
  • various tag sequences HA, myc, FLAG, His tag, etc.
  • the gp17 gene region into which a gene such as fluorescent protein is inserted is a region in which the expression of the gene such as fluorescent protein is functionally regulated by the promoter or regulatory region of the gp17 gene and the gp17 gene can be functionally expressed. Any area may be used as long as it is present. It is preferably in the coding region or in the transcription region near the end of the coding region of the gp17 gene, in which the function of the gp17 gene is less likely to be deleted, and more preferably at the 5'end or 3'end of the coding region of the gp17 gene.
  • the gp17 gene and a gene such as a fluorescent protein need to be linked so that the same promoter or regulatory region can control both genes, and may be linked via a spacer sequence or a linker sequence.
  • a fusion protein containing an amino acid sequence encoded by a gene such as gp17 gene and a fluorescent protein is expressed, once transcription is started by the promoter, transcription is performed through the coding region to the stop codon.
  • Genes such as gp17 gene and fluorescent protein exist in the 5' ⁇ 3'direction.
  • a gene recombination operation method for inserting a heterologous gene such as a fluorescent protein into a specific region of the phage genome is well known to those skilled in the art, and homologous recombination and genome editing techniques can be used.
  • the principle of the method of recombination operation is to prepare a DNA fragment containing a fragment to be incorporated and two recombination arms, for example by PCR amplification. These arms are homologous to the region adjacent to the gene to be inserted.
  • These DNA fragments can also be produced from nucleotides having a sequence of several tens of bases by PCR using primers containing homologous arms.
  • the recombinant T7 phage of the present invention can be used to prepare a phage display library for selection or screening well known in the art.
  • a nucleic acid molecule encoding a peptide having a random sequence is inserted into the gp10 gene region of the bacteriophage T7 genome in a state where the random peptide and capsid protein are expressed as a fusion protein, and the recombinant bacteriophage T7 is randomly expressed.
  • Each phage particle presents one polypeptide, and then the phage particle is selected based on its affinity interaction with the target, which is the molecule of interest.
  • Expression can be peptide is one per phage, but be used because it is possible to prepare the phage at a concentration of 10 12 / ml or more, the phage population that expressed various types of peptides as a library it can.
  • GTAGGTCAGGCCGTTGTGG SEQ ID NO: 1
  • 3'Primer GTTCTCTTCACGTGTCCTTGGGTACAG SEQ ID NO: 3
  • Each PCR product was subjected to agarose gel electrophoresis to cut out the corresponding band, and was eluted and purified using Gel Extension Kit.
  • the 5'and 3'ends of the purified 2.9 kb fragment were digested with restriction enzymes SfiI and PmlI, subjected to agarose gel electrophoresis again (results are shown in FIG. 4), and eluted and purified using Gel Extension Kit. did.
  • the infected BL21 was mixed with TOP agarose and sown on an LB plate, and it was confirmed by visual observation with a stereomicroscope that the plaques that appeared fluoresce. This stereomicrograph is shown in FIG.
  • the collected plaque is incubated with a phage extraction buffer (20 mM Tris-HCl, 100 mM NaCl, 6 mM sulfonyl 4 , pH 8.0) (4 ° C., 3 hours), mixed with a small amount of chloroform by inversion, and then centrifuged (3,000 ⁇ g). , 5 minutes), and the supernatant was collected to obtain a phage extract.
  • T7gp17-sfGFP phage genomic DNA was extracted by 2% SDS treatment, and the supernatant was centrifuged at 16,500 ⁇ g.
  • T7gp17-sfGFP phage genomic DNA (38.1 kb) was purified using QIAGEN).
  • the obtained phage genomic DNA was precipitated with ethanol and then digested with EcoRI and HindIII to obtain 21.5 kbp and 16.6 kbp fragments, subjected to 0.8% agarose gel electrophoresis and purified using QIAGEN GEL EXTRATION KIT. (Fig. 8).
  • the 5'and 3'ends of this PCR product were digested with EcoRI and HindIII and then purified by 2% agarose electrophoresis and QIAGEN GEL EXTRATION KIT.
  • the obtained insert DNA and 21.5 kbp And 16.6 kbp phage genomic DNA was subjected to a ligation reaction with T4 ligation, the ligation product was mixed with T7 Packing Exects (Merck Millipore), and an In vitro packing reaction was carried out.
  • the packing product was infected with Escherichia coli BL21.
  • T7gp17-sfGFP random peptide library (T7-sfGFP ⁇ 12) was dropped onto the nasal mucosa of anesthetized mice, and after a certain period of time, the brain was removed, homogenized and infected with Escherichia coli BL21, and spread on an LB plate (150 mm). After amplification (37 ° C., 2.5 hours), phage was recovered from the plaque. Using this phage, the process proceeded to the next round, and the same amount of phage as in Round 1 was dropped onto the nasal mucosa of the mouse, and the same operation as described above was repeated 4 times. As the round progressed, the “output / input” ratio of the recovered phage increased about 900-fold (Fig. 12).
  • Non-Patent Document 2 a fusion protein of fluorescent protein and T7 capsid protein is added to phage by overexpression by Escherichia coli using a helper plasmid, but in this method, wild-type T7 capsid protein is further excessively excessive. Simultaneous expression in E. coli is essential for phage production, which actually results in only a portion of all phages containing the fluorescent protein (6% -16%). That is, using this method, even if the positive phage group selected by screening is replicated and amplified for the next screening round, only a part (6% to 16%) of all phages are fluorescent.

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Abstract

The present invention addresses the issue of providing: a recombinant T7 phage having a gene inserted into a T7 phage genome such that the gene can be expressed, said gene being for a fluorescent protein, which is a labeling protein, or for an enzyme that is a color reaction catalyst; a random peptide phage library for a recombinant T7 phage; and a method for using same. The present invention inserts a gene for a fluorescent protein or a gene for an enzyme that is a color reaction catalyst, into a gp17 genetic region of a T7 phage such that the gene can be expressed, and creates a recombinant T7 phage that stably expresses, in a caudal section, a certain number of fluorescent proteins or enzymes that are catalysts for a color reaction. This random peptide phage library for recombinant T7 phages can be used for in vitro and in vivo screening.

Description

新規組換えバクテリオファージNewly recombinant bacteriophage
　本発明は、バクテリオファージＴ７の組換えバクテリオファージに関する。 The present invention relates to a recombinant bacteriophage of bacteriophage T7.
　バクテリオファージＴ７(Ｔ７ファージ)は、ポドウイルス科に属するエンベロープを持たないＤＮＡウイルスで、正２０面体のカプシドの内部に、直鎖状の二本鎖ＤＮＡをゲノムとして持つファージである。宿主細胞の大腸菌に感染してファージＤＮＡを宿主中で複製、増殖させて多数のファージ粒子を形成し、宿主の細胞膜を破り溶菌する。ゲノムは１９８３年に解読されており、約４０ｋｂｐの核酸配列からなり、５５の遺伝子を含む。 Bacteriophage T7 (T7 phage) is a non-enveloped DNA virus belonging to the family Podoviridae, and is a phage having a linear double-stranded DNA as a genome inside a regular icosaheda capsid. It infects Escherichia coli in the host cell and replicates and proliferates phage DNA in the host to form a large number of phage particles, breaking the cell membrane of the host and lysing. The genome was decoded in 1983 and consists of a nucleic acid sequence of about 40 kbp and contains 55 genes.
　大腸菌に感染するファージは、ファージディスプレイ法に利用されており、ファージの表面にファージのコートタンパク質と融合した外来タンパク質、ペプチドを、他の分子と相互作用できる形で提示することができるため、機能モチーフの探索のための最も有力な方法の一つとなっている。しかし、機能モチーフのスクリーニング過程におけるファージの検出工程に、ＥＬＩＳＡなどの煩雑な方法を用いるため、スクリーニングに莫大な手間と時間を要するという欠点がある。 Phage that infects E. coli is used in the phage display method, and functions because foreign proteins and peptides fused with the coat protein of the phage can be presented on the surface of the phage in a form that can interact with other molecules. It has become one of the most powerful methods for exploring motifs. However, since a complicated method such as ELISA is used in the phage detection step in the functional motif screening process, there is a drawback that the screening requires enormous labor and time.
　最近、ファージのイメージングのために、ＧＦＰなどの蛍光タンパク質を用いて、Ｔ７ファージ(非特許文献１、２)、Ｔ４ファージ(非特許文献３)、またはラムダファージ(非特許文献４)を可視化して検出する報告がなされている。しかし、ファージに発現させる蛍光タンパク質の数が１～３個と極めて少ないため、蛍光強度が弱く、また、ファージ当たりの蛍光タンパク質の数が制御できないため一定でなく、蛍光強度を指標にした正確なスクリーニングや検出をすることができない。 Recently, for imaging of phages, T7 phages (Non-Patent Documents 1 and 2), T4 phages (Non-Patent Documents 3), or lambda phage (Non-Patent Documents 4) have been visualized using fluorescent proteins such as GFP. There is a report to detect it. However, since the number of fluorescent proteins expressed on the phage is extremely small, 1 to 3, the fluorescence intensity is weak, and the number of fluorescent proteins per phage cannot be controlled, so that it is not constant and is accurate using the fluorescence intensity as an index. Cannot be screened or detected.
　非特許文献１および２には、ＧＦＰ－キャプシド融合タンパク質を発現するヘルパープラスミドを、大腸菌内で発現させてファージに補うこと、あるいは、ファージゲノムのキャプシドタンパクのプロモーター活性を極めて減弱させたＴ７ファージに、ＧＦＰ－キャプシド融合タンパク質遺伝子を組み込んだ上で、野生型キャプシドタンパクをヘルパープラスミドにより大腸菌内で過剰に発現させてファージに補うことにより、いずれの場合も、Ｔ７ファージ１分子あたりＧＦＰタンパク質を１個～３個含むＴ７ファージが得られたことが報告されている。しかし、この蛍光は極めて弱いものであり、また、発現させるＧＦＰの数を制御できないため、ファージによってＧＦＰの数が一定しておらず、蛍光強度を指標にしたスクリーニングを行うことができない。 In Non-Patent Documents 1 and 2, a helper plasmid expressing a GFP-capsid fusion protein is expressed in Escherichia coli to supplement the phage, or a T7 phage in which the promoter activity of the capsid protein in the phage genome is extremely attenuated. , GFP-capsid fusion protein gene is incorporated, and wild-type capsid protein is overexpressed in Escherichia coli by a helper plasmid to supplement the phage. In each case, one GFP protein is produced per molecule of T7 phage. It has been reported that T7 phage containing ~ 3 was obtained. However, since this fluorescence is extremely weak and the number of GFPs to be expressed cannot be controlled, the number of GFPs is not constant depending on the phage, and screening using the fluorescence intensity as an index cannot be performed.
　非特許文献３には、Ｔ４ファージキャプシドのアクセサリータンパクＨｏｃにＧＦＰを融合させたタンパクを、ヘルパープラスミドを用いた大腸菌による過剰発現でＴ４ファージに付加することにより、蛍光ファージにしたことが記載されているが、ファージに結合しなかった遊離のＨｏｃ－ＧＦＰタンパク質をＦＰＬＣで分離する必要があり、しかも、得られた蛍光Ｔ４ファージは、ＧＦＰのついていないＴ４ファージに比べ、１.１倍程度の蛍光強度しか有さない。
　非特許文献４には、λファージのネック部分にＧＦＰタンパクが取り込まれるように設計することが記載されているが、ファージに発現させる蛍光タンパク質の数が少なく、ファージ当たりの蛍光タンパク質の数が制御不能である点で変わらない。 Non-Patent Document 3 describes that a protein obtained by fusing GFP with the accessory protein Hoc of the T4 phage capside was added to the T4 phage by overexpression by Escherichia coli using a helper plasmid to obtain a fluorescent phage. However, it is necessary to separate the free Hoc-GFP protein that did not bind to the phage by FPLC, and the obtained fluorescent T4 phage has about 1.1 times the fluorescence of the T4 phage without GFP. It has only strength.
Non-Patent Document 4 describes that the design is such that the GFP protein is incorporated into the neck portion of the λ phage, but the number of fluorescent proteins expressed on the phage is small and the number of fluorescent proteins per phage is controlled. It does not change in that it is impossible.
　さらに、非特許文献１～３に記載されたファージは、蛍光タンパク質をキャプシドに融合させているために、ファージディスプレイ法に使用した場合に、蛍光タンパク質が、提示するタンパク質と立体障害を起こすという欠点がある。同様に、特許文献１に記載のルシフェラーゼ酵素遺伝子をキャプシド表面に発現させる組換えバクテリオファージにおいても、このような立体障害を起こす可能性がある。 Furthermore, since the phages described in Non-Patent Documents 1 to 3 have a fluorescent protein fused to a capsid, there is a drawback that the fluorescent protein causes steric damage to the presented protein when used in the phage display method. There is. Similarly, a recombinant bacteriophage expressing the luciferase enzyme gene described in Patent Document 1 on the surface of a capsid may cause such steric hindrance.
特表２０１７－５１１７０２号公報Special Table 2017-511702
　本発明は、Ｔ７ファージを十分に可視化することのできる強度で、活性を有する蛍光タンパク質を発現することができ、あるいは、Ｔ７ファージを可視化できる、活性を有する呈色反応を触媒する酵素を発現することのできる、ファージディスプレイ法に好適に使用できる組換えＴ７ファージを提供することをその課題とする。
　また、本発明は、そのような組換えＴ７ファージをファージディスプレイ法に使用する方法を提供することを課題とする。 The present invention expresses an enzyme that catalyzes an active color reaction that can express an active fluorescent protein or can visualize a T7 phage with a strength capable of sufficiently visualizing the T7 phage. It is an object of the present invention to provide recombinant T7 phage which can be suitably used for the phage display method.
Another object of the present invention is to provide a method for using such recombinant T7 phage in the phage display method.
　本発明者は、上記課題を解決すべく研究を重ねた結果、標識タンパク質である蛍光タンパク質の遺伝子または呈色反応を触媒する酵素の遺伝子を、Ｔ７ファージの特定の遺伝子領域に発現可能に挿入することにより、Ｔ７ファージが宿主に感染しその複製中に、蛍光タンパク質または呈色反応を触媒する酵素が正しく折り畳まれ、活性を有する立体構造でファージタンパク質の一部として発現されることを見出し、本発明の完成に至った。この組換えＴ７ファージは、このように異種遺伝子を発現できるだけでなく、溶菌後感染性ウイルスとして新たな宿主に感染することができる。 As a result of repeated studies to solve the above problems, the present inventor inserts a gene for a fluorescent protein, which is a labeling protein, or a gene for an enzyme that catalyzes a coloring reaction into a specific gene region of a T7 phage so as to be expressible. As a result, we found that during the replication of T7 phage by infecting the host, the fluorescent protein or the enzyme that catalyzes the coloring reaction is correctly folded and expressed as a part of the phage protein in an active three-dimensional structure. The invention was completed. Not only can this recombinant T7 phage express a heterologous gene in this way, but it can also infect a new host as a post-lytically infectious virus.
　すなわち、本発明は、以下の(１)～(７)の組換えバクテリオファージＴ７に関する。
（１）組換えバクテリオファージＴ７であって、バクテリオファージＴ７のゲノムのｇｐ１７遺伝子領域に、標識タンパク質の遺伝子が発現可能に挿入されていることを特徴とする、組換えバクテリオファージＴ７。
（２）前記標識タンパク質が、蛍光タンパク質または発光または呈色反応を触媒する酵素である、上記(１)に記載の組換えバクテリオファージＴ７。
（３）前記蛍光タンパク質が緑色蛍光タンパク質(ＧＦＰ)であり、前記呈色反応を触媒する酵素が、ルシフェラーゼ、β－グルクロニダーゼ(ＧＵＳ)、β－ガラクトシダーゼ、西洋ワサビペルオキシダーゼのいずれか一つである、上記(２)に記載の組換えバクテリオファージＴ７。
（４）前記標識タンパク質の遺伝子が、ｇｐ１７遺伝子コード領域末端近傍に挿入されている、上記(１)に記載の組換えバクテリオファージＴ７。
（５）前記ｇｐ１７遺伝子コード領域末端近傍が、３´末端領域である、上記(４)に記載の組換えバクテリオファージＴ７。
（６）組換えバクテリオファージＴ７が、宿主細胞に感染後の細胞質におけるファージ複製の間に、前記蛍光タンパク質の遺伝子または発光または呈色反応を触媒する酵素の遺伝子を発現して、活性を有する蛍光タンパク質または発光または呈色反応を触媒する酵素生成物を生じる、上記(２)に記載の組換えバクテリオファージＴ７。
（７）前記蛍光タンパク質または発光または呈色反応を触媒する酵素が、バクテリオファージＴ７の尾部に発現する、上記(６)に記載の組換えバクテリオファージＴ７。 That is, the present invention relates to the following recombinant bacteriophage T7 (1) to (7).
(1) Recombinant bacteriophage T7, wherein the gene of the labeled protein is expressively inserted into the gp17 gene region of the genome of the recombinant bacteriophage T7.
(2) The recombinant bacteriophage T7 according to (1) above, wherein the labeled protein is a fluorescent protein or an enzyme that catalyzes a luminescence or color reaction.
(3) The fluorescent protein is green fluorescent protein (GFP), and the enzyme that catalyzes the coloring reaction is one of luciferase, β-glucuronidase (GUS), β-galactosidase, and horseradish peroxidase. The recombinant bacterophage T7 according to (2) above.
(4) The recombinant bacteriophage T7 according to (1) above, wherein the gene of the labeled protein is inserted near the end of the gp17 gene coding region.
(5) The recombinant bacteriophage T7 according to (4) above, wherein the vicinity of the terminal of the gp17 gene coding region is the 3'terminal region.
(6) Recombinant bacteriophage T7 expresses the gene of the fluorescent protein or the gene of an enzyme that catalyzes a luminescence or coloring reaction during phage replication in the cytoplasm after infection of the host cell, and has activity. The recombinant bacteriophage T7 according to (2) above, which produces a protein or an enzyme product that catalyzes a luminescence or color reaction.
(7) The recombinant bacteriophage T7 according to (6) above, wherein the fluorescent protein or an enzyme that catalyzes a luminescence or color reaction is expressed in the tail of the bacteriophage T7.
　また、本発明は、以下(８)の組換えバクテリオファージＴ７のランダムペプチドファージライブラリー、または(９)、(１０)のランダムペプチドファージライブラリーを使用する方法に関する。
（８）組換えバクテリオファージＴ７であって、バクテリオファージＴ７のゲノムのｇｐ１７遺伝子領域に標識タンパク質の遺伝子が発現可能に挿入されている組換えバクテリオファージＴ７のゲノムのｇｐ１０遺伝子領域に、ランダムペプチドをコードする核酸分子を挿入した、組換えバクテリオファージＴ７のランダムペプチドファージライブラリー。
（９）上記（８）に記載の組換えバクテリオファージＴ７のランダムペプチドファージライブラリーを使用するｉｎ　ｖｉｔｒｏスクリーニング方法。
（１０）上記（８）に記載の組換えバクテリオファージＴ７のランダムペプチドファージライブラリーを使用するｉｎ　ｖｉｖｏスクリーニング方法。 The present invention also relates to a method using the random peptide phage library of recombinant bacteriophage T7 described in (8) below or the random peptide phage library of (9) and (10) below.
(8) In recombinant bacteriophage T7, a random peptide is placed in the gp10 gene region of the genome of recombinant bacteriophage T7 in which the gene of the labeled protein is expressively inserted into the gp17 gene region of the genome of bacteriophage T7. A random peptide phage library of recombinant bacteriophage T7 with the encoding nucleic acid molecule inserted.
(9) An in vitro screening method using the random peptide phage library of recombinant bacteriophage T7 according to (8) above.
(10) An in vivo screening method using the random peptide phage library of recombinant bacteriophage T7 according to (8) above.
　従来法では、１ファージ分子あたりの発現される蛍光タンパク質が１～３個までと少ないが、本発明の組換えファージでは、従来法に比べ１オーダー上の多数の蛍光タンパク質が発現するため、蛍光強度に各段の差がある。
　また、従来法では、ファージ分子あたりの蛍光タンパク質の数を制御することができず、ファージにより蛍光タンパクの数が異なり、蛍光強度が一定ではないが、本発明の組換えファージには、すべてのファージに必ず同じ数の蛍光タンパク質が発現するため、正確な一定の蛍光強度によるスクリーニングを実現できる。 In the conventional method, the number of fluorescent proteins expressed per phage molecule is as small as 1 to 3, but in the recombinant phage of the present invention, a large number of fluorescent proteins expressed by one order higher than in the conventional method are expressed, so that fluorescence is achieved. There is a difference in strength at each stage.
Further, in the conventional method, the number of fluorescent proteins per phage molecule cannot be controlled, the number of fluorescent proteins differs depending on the phage, and the fluorescence intensity is not constant. Since the same number of fluorescent proteins are always expressed in the phage, screening with an accurate constant fluorescence intensity can be realized.
　さらに、ファージにヘルパープラスミドを用いて大腸菌で蛍光タンパク質を過剰発現させて補充する必要がないので、スクリーニングにおけるパニングの過程で、選択したファージを複製・増殖するラウンドごとに、未結合の蛍光タンパク質と目的の蛍光を発するファージとを毎回分離する手間もかからず、また、選択増幅したすべてのファージに蛍光タンパク質が間違いなく結合しているかを、ラウンドのたびに検証する必要がない。
　また、本発明の組換えファージをファージディスプレイに使用する場合に、ライブラリー部分のランダムペプチドを発現するキャプシド部分とは異なる構造部分に蛍光タンパク質が発現することから、ターゲットに対する結合の立体障害を生じることなく、ファージディスプレイ法で重要な要素であるターゲットとの結合に対しても見逃しが起きないという優れた効果を奏する。 Furthermore, since it is not necessary to overexpress and supplement the fluorescent protein in E. coli using a helper plasmid in the phage, the unbound fluorescent protein is added to each round in which the selected phage is replicated and proliferated during the panning process in the screening. There is no need to separate the phages that emit the desired fluorescence each time, and it is not necessary to verify that the fluorescent protein is definitely bound to all the selectively amplified phages in each round.
In addition, when the recombinant phage of the present invention is used for phage display, the fluorescent protein is expressed in a structural portion different from the capsid moiety expressing the random peptide in the library portion, which causes a steric disorder of binding to the target. It has an excellent effect that it does not overlook the binding to the target, which is an important element in the phage display method.
制限酵素ＳｆｉＩおよびＰｍｌＩによるＴ７ファージゲノムＤＮＡの切断部位を示す。The cleavage sites of T7 phage genomic DNA by restriction enzymes SfiI and PmlI are shown. 制限酵素で切断後のＤＮＡフラグメントのアガロースゲル電気泳動の結果を示す。The results of agarose gel electrophoresis of the DNA fragment after cleavage with a restriction enzyme are shown. ＧＳスペーサーとｓｆＧＦＰ遺伝子を含む２.９ｋｂフラグメントの調製方法を示す。A method for preparing a 2.9 kb fragment containing a GS spacer and the sfGFP gene is shown. ２.９ｋｂフラグメントのアガロースゲル電気泳動の結果を示す。The results of agarose gel electrophoresis of the 2.9 kb fragment are shown. 組換えＴ７ｇｐ１７－ｓｆＧＦＰのゲノムの概略を示す。The outline of the genome of recombinant T7gp17-sfGFP is shown. 組換えＴ７ｇｐ１７－ｓｆＧＦＰを感染させた大腸菌のプラークの実体蛍光顕微鏡写真を示す。FIG. 3 shows a solid fluorescence micrograph of a plaque of Escherichia coli infected with recombinant T7gp17-sfGFP. 第２世代の組み換えファージのｇｐ１７－ｓｆＧＦＰのウエスタンブロットを示す。A Western blot of gp17-sfGFP of second generation recombinant phage is shown. Ｔ７ｇｐ１７－ｓｆＧＦＰファージゲノム全長ＤＮＡと、制限酵素で切断後のＤＮＡフラグメントのアガロースゲル電気泳動の結果を示す。The results of agarose gel electrophoresis of the T7gp17-sfGFP phage genome full-length DNA and the DNA fragment cleaved with a restriction enzyme are shown. 動物細胞発現用プラスミドベクターによるｍＬＤＬＲｅｘｔ－ｍｙｃの発現を示す抗ｍｙｃ抗体を用いたウエスタンブロット。Western blot using an anti-myc antibody showing the expression of mLDLRext-myc by a plasmid vector for animal cell expression. 実施例５におけるラウンド１→３でのＬＤＬＲｅｘｔ結合性を示すファージの占める割合の変化を示す。The change in the proportion of phage showing LDLRext binding property in rounds 1 → 3 in Example 5 is shown. 実施例５におけるラウンド３のファージが、ｍＬＤＬＲ－ｍＣｈｅｒｒｙ発現細胞に特異的に結合することを示す、蛍光顕微鏡写真。(ａ、ＧＦＰシグナル（緑）；ｂ、ｍＣｈｅｒｒｙシグナル(赤)；ｃ、ａとｂの重ね合わせ)Fluorescence micrographs showing that round 3 phage in Example 5 specifically binds to mLDLR-mCherry expressing cells. (a, GFP signal (green); b, mCherry signal (red); superposition of c, a and b) 実施例６におけるラウンド１→４でのマウス脳への侵入機能を示すファージの占める割合の変化を示す。The change in the proportion of phage showing the function of invading the mouse brain in Rounds 1 → 4 in Example 6 is shown. 実施例６でのファージの有するペプチドを末端ＴＡＭＲＡ化し、マウスの鼻粘膜に滴下し一定時間後、頭蓋骨を落射型蛍光実体顕微鏡により観察した写真を示す。A photograph of the peptide possessed by the phage in Example 6 converted to terminal TAMRA, dropped onto the nasal mucosa of a mouse after a certain period of time, and the skull observed with an epi-illumination stereomicroscope is shown. 図１３の頭蓋骨の凍結切片のＴＡＭＲＡシグナルを、落射型蛍光顕微鏡により観察した写真を示す。スケールバー：50μm。The photograph which observed the TAMRA signal of the frozen section of the skull of FIG. 13 by the epi-illumination fluorescence microscope is shown. Scale bar: 50 μm.
　バクテリオファージＴ７(Ｔ７ファージ)は、二本鎖直鎖状のゲノムＤＮＡを持つ溶菌性のファージであり、大腸菌表面にあるＬＰＳに結合して、大腸菌内へＤＮＡを注入して感染する。ゲノムＤＮＡを格納するファージの頭部は、４１５個のＧ１０タンパク質の会合により形成され、その下に尾部が付加されている。 Bacteriophage T7 (T7 phage) is a lytic phage having double-stranded linear genomic DNA, binds to LPS on the surface of Escherichia coli, and injects DNA into Escherichia coli to infect. The head of the phage that stores the genomic DNA is formed by the association of 415 G10 proteins, and the tail is added below it.
　ゲノムＤＮＡは、約４０ｋｂｐの核酸配列からなり５５の遺伝子を含み、必須遺伝子には整数の番号が付けられている。ｇｐ１はＤＮＡ依存性ＲＮＡポリメラーゼ遺伝子で、感染初期に大腸菌由来ＲＮＡポリメラーゼによって転写された後、それ以降のウイルス遺伝子の転写を行い、ｇｐ５はＤＮＡポリメラーゼ遺伝子でＤＮＡの複製を行う。ｇｐ１０は頭部キャプシドの主成分である外郭を構成する遺伝子であり、ｇｐ１７は尾部の付け根から伸びる尾部繊維タンパク質遺伝子で、尾部繊維タンパク質は３分子で一本の繊維を形成し、この繊維が６本伸びて尾部の脚を形成する。 Genomic DNA consists of a nucleic acid sequence of about 40 kbp and contains 55 genes, and essential genes are numbered with integers. gp1 is a DNA-dependent RNA polymerase gene, which is transcribed by E. coli-derived RNA polymerase in the early stage of infection, and then the viral gene is transcribed, and gp5 is a DNA polymerase gene that replicates DNA. gp10 is a gene that constitutes the outer shell, which is the main component of the head capsid, gp17 is a tail fiber protein gene that extends from the base of the tail, and tail fiber protein forms one fiber with 3 molecules, and this fiber is 6 It stretches to form the tail leg.
　本発明において、「バクテリオファージ」もしくは「ファージ」とは、それらを複製する手段として、天然において細菌を使用するように進化したウイルスである。ファージは、このことを、ファージ自体を細菌に付着させ、そのＤＮＡをその細菌の中に注入し、上記ファージを数百倍もしくはさらには数千倍も複製するようにその細菌を誘導することによって行う。これをファージ増幅ともいう。 In the present invention, "bacteriophage" or "phage" is a virus that has evolved to use bacteria in nature as a means of replicating them. The phage attaches the phage itself to the bacterium, injects its DNA into the bacterium, and induces the bacterium to replicate hundreds or even thousands of times the phage. Do. This is also called phage amplification.
　本発明において、「バクテリオファージＴ７」とは、天然のバクテリオファージＴ７と少なくとも９９％、９８％、９７％、９６％、９５％、９４％、９３％、９２％、９１％、９０％、８９％、８８％、８７％、８６％、８５％、８４％、８３％、８２％、８１％、８０％、７９％、７８％、７７％、７６％、７５％の相同性を有するゲノムを有し、かつ上記ファージ増幅を行い、最終的に細菌の細胞壁を破り(溶菌)、細胞外に放出されるバクテリオファージをいう。
　また、「組換え」とは、他の方法では見いだされない遺伝的物質を一緒にするための遺伝的な改変をいう。 In the present invention, "bacteriophage T7" is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89 with natural bacteriophage T7. %, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75% homologous genomes A bacteriophage that has and performs the above-mentioned phage amplification, finally breaks the cell wall of the bacterium (lysis), and is released to the outside of the cell.
Also, "recombination" refers to a genetic modification to bring together genetic material that is not otherwise found.
　本発明の組換えファージに導入される標識タンパク質の遺伝子としては、基質が必要ない点で蛍光タンパク質の遺伝子が好ましい。蛍光タンパク質としては、市販されている緑色蛍光タンパク質(ＧＦＰ)、赤色蛍光タンパク質(ＲＦＰ)、黄色蛍光タンパク質(ＹＦＰ)、またはシアン蛍光タンパク質(ＣＦＰ)の遺伝子を用いることができ、これらのタンパク質の修飾型のすべてのｖａｒｉａｎｔのいずれの遺伝子も用いることができる。 As the gene for the labeled protein introduced into the recombinant phage of the present invention, the gene for the fluorescent protein is preferable because it does not require a substrate. As the fluorescent protein, commercially available green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), or cyanide fluorescent protein (CFP) genes can be used, and modifications of these proteins can be used. Any gene of any variant of type can be used.
　また、発光または呈色反応を触媒する酵素の遺伝子を用いることができ、たとえば、ルシフェラーゼ(Ｌｕｃ)、β－グルクロニダーゼ(ＧＵＳ)、β－ガラクトシダーゼ(Ｇａｌ)、西洋ワサビペルオキシダーゼ(ＨＲＰ)、アルカリホスファターゼ(ＡＰ)等の市販されている遺伝子が挙げられる。 In addition, genes of enzymes that catalyze luminescence or color reaction can be used, for example, luciferase (Luc), β-glucuronidase (GUS), β-galactosidase (Gal), horseradish peroxidase (HRP), alkaline phosphatase ( Examples include commercially available genes such as AP).
　また、蛍光・発光タンパク質や蛍光色素に対して結合性を有するペプチドをコードするＤＮＡを用いることができ、それ以外にも、ビオチン化タグ配列ペプチド(大腸菌のビオチンライゲースＢｉｒＡのアクセプターペプチド)、あるいは各種タグ配列(ＨＡ、ｍｙｃ、ＦＬＡＧ、Ｈｉｓタグ等)も用いることができる。 In addition, DNA encoding a peptide having a binding property to a fluorescent / photoprotein or a fluorescent dye can be used. In addition, a biotinylated tag sequence peptide (acceptor peptide of Escherichia coli biotin-ligase BirA), Alternatively, various tag sequences (HA, myc, FLAG, His tag, etc.) can also be used.
　本発明において、「ｇｐ１７遺伝子領域に、標識タンパク質の遺伝子が発現可能に挿入されている」とは、両遺伝子が発現する際に、ｇｐ１７遺伝子の発現産物である尾部繊維タンパク質と蛍光タンパク質等とが融合タンパク質として発現される状態で、ｇｐ１７遺伝子領域に標識タンパク質の遺伝子が挿入されている状態を意味する。ｇｐ１７遺伝子は、尾部の付け根から伸びる尾部繊維タンパク質遺伝子であり、３分子で一本の繊維を形成する。Ｔ７ファージはその繊維からなる脚を６本有するため、ｇｐ１７遺伝子領域に蛍光タンパク質等の遺伝子を発現可能に挿入することにより、３×６＝１８の１８個の蛍光タンパク質、酵素、タグ配列等が脚表面に発現した尾部を有する組換えファージを作成することができる。 In the present invention, "the gene of the labeled protein is expressively inserted into the gp17 gene region" means that when both genes are expressed, the tail fiber protein, the fluorescent protein, etc., which are the expression products of the gp17 gene, are used. It means a state in which the gene of the labeled protein is inserted into the gp17 gene region in a state of being expressed as a fusion protein. The gp17 gene is a tail fiber protein gene that extends from the base of the tail, and three molecules form one fiber. Since T7 phage has 6 legs consisting of its fibers, 18 fluorescent proteins, enzymes, tag sequences, etc. of 3 × 6 = 18 can be obtained by inserting genes such as fluorescent proteins into the gp17 gene region so that they can be expressed. Recombinant phage having a tail expressed on the leg surface can be produced.
　蛍光タンパク質等の遺伝子が挿入されるｇｐ１７遺伝子領域は、蛍光タンパク質等の遺伝子の発現が、ｇｐ１７遺伝子のプロモーターまたは調節領域により機能的に制御され、かつ、ｇｐ１７遺伝子が機能的に発現可能な領域であればいかなる領域であってもよい。ｇｐ１７遺伝子の機能が欠失しにくいｇｐ１７遺伝子のコード領域の末端近傍のコード領域内または転写領域内が好ましく、ｇｐ１７遺伝子のコード領域の５´末端または３´末端がより好ましい。 The gp17 gene region into which a gene such as fluorescent protein is inserted is a region in which the expression of the gene such as fluorescent protein is functionally regulated by the promoter or regulatory region of the gp17 gene and the gp17 gene can be functionally expressed. Any area may be used as long as it is present. It is preferably in the coding region or in the transcription region near the end of the coding region of the gp17 gene, in which the function of the gp17 gene is less likely to be deleted, and more preferably at the 5'end or 3'end of the coding region of the gp17 gene.
　ｇｐ１７遺伝子と蛍光タンパク質等の遺伝子は、同じプロモーターまたは調節領域が、両遺伝子を制御できるように連結される必要があり、スペーサー配列またはリンカー配列を介して連結してもよい。ｇｐ１７遺伝子と蛍光タンパク質等の遺伝子によりコードされるアミノ酸配列を含む融合タンパク質が発現するように、インフレームで連結させることにより、一旦プロモーターで転写が始まると、コード領域を通って終止コドンまで転写が続く。ｇｐ１７遺伝子および蛍光タンパク質等の遺伝子は、５´→３´方向に存在する。 The gp17 gene and a gene such as a fluorescent protein need to be linked so that the same promoter or regulatory region can control both genes, and may be linked via a spacer sequence or a linker sequence. By linking in-frame so that a fusion protein containing an amino acid sequence encoded by a gene such as gp17 gene and a fluorescent protein is expressed, once transcription is started by the promoter, transcription is performed through the coding region to the stop codon. Continue. Genes such as gp17 gene and fluorescent protein exist in the 5'→ 3'direction.
　蛍光タンパク質等の異種遺伝子をファージゲノムの特定の領域に挿入する遺伝子組換え操作方法は、当業者に周知であり、相同組み換えやゲノム編集技術を用いることができる。組換え操作の方法の原則は、たとえばＰＣＲ増幅によって、組み込むべき断片及び２つの組換えアームを含むＤＮＡ断片を作製することである。これらのアームは、挿入する遺伝子に隣接した領域に相同である。これらのＤＮＡ断片は、相同なアームを含んだプライマーを用いたＰＣＲによって、数十塩基配列のヌクレオチドから作り出すことも可能である。 A gene recombination operation method for inserting a heterologous gene such as a fluorescent protein into a specific region of the phage genome is well known to those skilled in the art, and homologous recombination and genome editing techniques can be used. The principle of the method of recombination operation is to prepare a DNA fragment containing a fragment to be incorporated and two recombination arms, for example by PCR amplification. These arms are homologous to the region adjacent to the gene to be inserted. These DNA fragments can also be produced from nucleotides having a sequence of several tens of bases by PCR using primers containing homologous arms.
　本発明では、Ｔ７ゲノムの全長ＤＮＡ中での切断部位がそれぞれ１か所である２つの制限酵素の制限部位が、Ｔ７ゲノムのｇｐ１７遺伝子の３´末端近傍のコード領域中と３´末端下流領域に存在するため、それらの制限酵素部位を利用して、ｇｐ１７遺伝子コード領域の３´末端にＧＦＰ遺伝子を導入することができる。 In the present invention, the restriction sites of the two restriction enzymes having one cleavage site in the full-length DNA of the T7 genome are in the coding region near the 3'end of the gp17 gene of the T7 genome and in the downstream region of the 3'end. Since it is present in, the GFP gene can be introduced into the 3'end of the gp17 gene coding region by utilizing those restriction enzyme sites.
　後述する実施例では、Ｔ７ファージとして、野生型のＴ７ファージ(４０ｋｂ)のキャプシドタンパク質のＣ末端に任意のペプチドを融合タンパク質として発現させることのできる改変体である、Ｔ７　Ｓｅｌｅｃｔ　４１５－１ｂ(Merk Millipore社製)を用いる。キャプシドタンパク質の発現に関わるＤＮＡ部分以外は、Ｔ７　Ｓｅｌｅｃｔ　４１５－１ｂのＤＮＡは、野生型Ｔ７ゲノムＤＮＡと同じである。
　ＳｆｉＩ－ＰｍｌＩ間のみならず、ｇｐ１７遺伝子を含んだ前後のゲノムはすべて保存されているので、本発明の組換えＴ７ファージの作成方法は、野生型Ｔ７を含めたすべてのＴ７改変体ファージ(Ｔ７　Ｓｅｌｅｃｔ　４１５－１ｂ，１０－３ｂ，１－１ｂ，１－２ａ，１－２ｂ，１－２ｃ：Merk Millipore社製)に適用することができる。 In the examples described later, as T7 phage, T7 Select 415-1b (Merk Millipore), which is a variant capable of expressing an arbitrary peptide as a fusion protein at the C-terminal of the capsid protein of wild T7 phage (40 kb). (Manufactured by the company) is used. The DNA of T7 Select 415-1b is the same as the wild-type T7 genomic DNA, except for the DNA portion involved in the expression of the capsid protein.
Since all the genomes before and after containing the gp17 gene are conserved as well as between SfiI and PmlI, the method for producing recombinant T7 phage of the present invention is used for all T7 mutant phage (T7) including wild-type T7. It can be applied to Select 415-1b, 10-3b, 1-1b, 1-2a, 1-2b, 1-2c: manufactured by Merck Millipore).
　最初に、Ｔ７ファージのゲノムＤＮＡを制限酵素ＳｆｉＩおよびＰｍｌＩにより切断し、得られた切断断片である３３.３ｋｂ、２.１ｋｂ、１.９ｋｂの３本のＤＮＡ断片を、アガロースゲル電気泳動によりサイズ分離して、３３.３ｋｂと１.９ｋｂのそれぞれのＤＮＡ断片のサイズのバンドのＤＮＡを溶出させてそれぞれ精製する。各精製ＤＮＡフラグメントは、アガロース電気泳動で精製をさらに確認する。 First, the genomic DNA of T7 phage was cleaved with restriction enzymes SfiI and PmlI, and the obtained cleaved fragments, 33.3 kb, 2.1 kb, and 1.9 kb, were sized by agarose gel electrophoresis. The DNA is separated and the DNA of the band of the size of each DNA fragment of 33.3 kb and 1.9 kb is eluted and purified respectively. Each purified DNA fragment is further confirmed for purification by agarose gel electrophoresis.
　次いで、Ｔ７ゲノムのｇｐ１７配列内のＳｆｉＩ制限部位からｇｐ１７下流にあるＰｍｌＩ制限部位までの上記２.１ｋｂのＤＮＡ断片において、ｇｐ１７コード領域の３´末端にＧＳスペーサー配列をコードする遺伝子を介してＧＦＰ遺伝子が連結されている約２.９ｋｂのフラグメントを、以下のオーバーラップ伸長ＰＣＲ法を用いて作製する。 Next, in the above 2.1 kb DNA fragment from the SfiI restriction site in the gp17 sequence of the T7 genome to the PmlI restriction site downstream of gp17, GFP was mediated at the 3'end of the gp17 coding region via the gene encoding the GS spacer sequence. A fragment of about 2.9 kb to which the genes are linked is prepared using the following overlap extension PCR method.
　このフラグメントを作製するために、まず２.９ｋｂを約１ｋｂずつの３本のフラグメント(以下、それぞれＡ、Ｂ、Ｃという。)に分けて、それぞれ化学合成により作製する。Ａ、Ｂ、Ｃのそれぞれの５´末端と３´末端に、３０ｂｐのオーバーラップ配列を付加する。フラグメントＡとＢのオーバーラップ伸長ＰＣＲにより、ＰＣＲ産物Ａ＋Ｂが作製され、このＡ＋Ｂ産物とフラグメントＣとのオーバーラップ伸長ＰＣＲにより、ＰＣＲ産物Ａ＋Ｂ＋Ｃという目的の２.９ｋｂ産物を得る。各ＰＣＲ産物はアガロースゲル電気泳動により分離して、該当するバンドを切り取り、精製した２.９ｋｂフラグメントの５´末端と３´末端を、制限酵素ＳｆｉＩおよびＰｍｌＩで消化する。 In order to prepare this fragment, first, 2.9 kb is divided into three fragments of about 1 kb each (hereinafter, referred to as A, B, and C, respectively), and each of them is prepared by chemical synthesis. A 30 bp overlap sequence is added to the 5'end and 3'end of A, B, and C, respectively. The PCR product A + B is prepared by the overlap extension PCR of fragments A and B, and the target 2.9 kb product of the PCR product A + B + C is obtained by the overlap extension PCR of the A + B product and the fragment C. Each PCR product is separated by agarose gel electrophoresis, the corresponding band is cut off, and the 5'end and 3'end of the purified 2.9 kb fragment are digested with restriction enzymes SfiI and PmlI.
　次に、Ｔ７ファージのゲノムＤＮＡを制限酵素ＳｆｉＩおよびＰｍｌＩにより切断して得られ、精製しておいた３３.３ｋｂと１.９ｋｂの２つのフラグメントと、オーバーラップ伸長ＰＣＲ産物である精製した２.９ｋｂフラグメントの合計３本のフラグメントを、Ｔ４ＤＮＡリガーゼを用いてライゲーション反応により連結する。このライゲーション反応産物とＴ７ファージ抽出物とを混合してパッケージング反応を行い、組換えＴ７ファージを作成して大腸菌に感染させる。 Next, the genomic DNA of T7 phage was cleaved with restriction enzymes SfiI and PmlI, and the two fragments of 33.3 kb and 1.9 kb, which were purified, and the purified product, which is an overlap extension PCR product, were purified. A total of 3 fragments of 9 kb fragments are ligated by ligation reaction using T4 DNA ligase. The ligation reaction product and the T7 phage extract are mixed and subjected to a packaging reaction to prepare recombinant T7 phage and infect Escherichia coli.
　感染させた大腸菌をＬＢプレートに蒔いて、出現するプラークが蛍光を発することにより、蛍光タンパク質が正確に折り畳まれて発現する組換えＴ７ファージを得られることを確認する。そして、この蛍光を発する組換えＴ７ファージの感染性を、さらに大腸菌に感染させて液体培地中で増殖させ、溶菌を確認してから溶菌物を遠心して、上清中のファージ液が蛍光を発することにより確認する。 Sow the infected Escherichia coli on an LB plate and confirm that the emerging plaque fluoresces to obtain recombinant T7 phage on which the fluorescent protein is accurately folded and expressed. Then, the infectivity of the recombinant T7 phage that emits fluorescence is further infected with Escherichia coli and propagated in a liquid medium, and after confirming the lysis, the lysate is centrifuged, and the phage solution in the supernatant fluoresces. Confirm by.
　本発明の組換えＴ７ファージは、１ファージ分子あたり１８個の蛍光タンパク質が発現するため、蛍光強度が強く、しかもすべてのファージに必ず同じ数の蛍光タンパク質が発現するため、正確な一定の蛍光強度によるスクリーニングを実現できる。 Since the recombinant T7 phage of the present invention expresses 18 fluorescent proteins per phage molecule, the fluorescence intensity is strong, and since the same number of fluorescent proteins are always expressed in all phages, an accurate constant fluorescence intensity is obtained. Screening can be realized.
　本発明の組換えＴ７ファージは、当技術分野において周知の選択またはスクリーニングのためのファージディスプレイライブラリーの作製に使用することができる。ランダムな配列を有するペプチドをコードする核酸分子を、バクテリオファージＴ７のゲノムのｇｐ１０遺伝子領域に、ランダムペプチドとキャプシドタンパク質とが融合タンパク質として発現される状態で挿入して、組換えバクテリオファージＴ７のランダムペプチドファージライブラリーを作成する。それぞれのファージ粒子は１つポリペプチドを提示し、次いでファージ粒子は、関心対象の分子であるターゲットとの親和性相互作用に基づいて選択する。発現できるペプチドは１ファージ当たり１種類であるが、１０１２個／ｍｌ以上の濃度でファージを調製することが可能であるため、さまざまな種類のペプチドを発現したファージ集団をライブラリーとして用いることができる。 The recombinant T7 phage of the present invention can be used to prepare a phage display library for selection or screening well known in the art. A nucleic acid molecule encoding a peptide having a random sequence is inserted into the gp10 gene region of the bacteriophage T7 genome in a state where the random peptide and capsid protein are expressed as a fusion protein, and the recombinant bacteriophage T7 is randomly expressed. Create a peptide phage library. Each phage particle presents one polypeptide, and then the phage particle is selected based on its affinity interaction with the target, which is the molecule of interest. Expression can be peptide is one per phage, but be used because it is possible to prepare the phage at a concentration of 10 12 / ml or more, the phage population that expressed various types of peptides as a library it can.
　本発明のファージディスプライブラリーを構成するファージは、蛍光を発するため、ファージライブラリーのｉｎ　ｖｉｔｒｏスクリーニングだけでなく、ｉｎ　ｖｉｖｏスクリーニングの解析工程も可視化でき、一定強度のファージの極めて迅速かつ正確な選択増幅によるスクリーニングを実現できることが特徴である。
　本発明の組換えＴ７ファージは、ライブラリー部分のランダムペプチドを発現する頭部キャプシド部分とは異なる尾部部分に、蛍光タンパク質が発現することから、ターゲットに対する結合の立体障害を生じることなく、ファージディスプレイ法で重要な要素であるターゲットとの結合の見逃しが起きないという顕著な効果を有する。 Since the phages constituting the phage disp library of the present invention fluoresce, not only the in vitro screening of the phage library but also the analysis process of in vivo screening can be visualized, and extremely rapid and accurate selection of phages of constant intensity can be performed. The feature is that screening by amplification can be realized.
Since the recombinant T7 phage of the present invention expresses a fluorescent protein in the tail portion different from the head capsid portion expressing the random peptide in the library portion, the phage display does not cause a steric disorder of binding to the target. It has a remarkable effect that the coupling with the target, which is an important element in the law, is not overlooked.
　以下、本発明を実施例に基づいて具体的に説明するが、本発明は、以下の実施例によって何ら限定されるものではない。 Hereinafter, the present invention will be specifically described based on examples, but the present invention is not limited to the following examples.
　Ｔ７　Ｓｅｌｅｃｔ　４１５－１ｂ　ＤＮＡを制限酵素ＳｆｉＩおよびＰｍｌＩ(New England Biolab社製)で切断して、３３.３ｋｂ、２.１ｋｂ、１.９ｋｂの３本のＤＮＡ断片を得た。概略を図１に示す。この３本のＤＮＡフラグメントをアガロースゲル電気泳動で分離し(結果を図２に示す。)、電気泳動後、ゲルから各フラグメントのそれぞれのバンドを切り出し、以下のように精製した。 T7 Select 415-1b DNA was cleaved with restriction enzymes SfiI and PmlI (manufactured by New England Biolabs) to obtain three DNA fragments of 33.3 kb, 2.1 kb and 1.9 kb. The outline is shown in FIG. These three DNA fragments were separated by agarose gel electrophoresis (results are shown in FIG. 2), and after electrophoresis, each band of each fragment was excised from the gel and purified as follows.
　３３.３ｋｂフラグメントは、ＧｅＢＡｆｌｅｘ－ｔｕｂｅ　Ｄｉａｌｙｓｉｓ　Ｋｉｔ(MW:3500、WACO社製)を用いて、切り出したゲル片から電気泳動による溶出(１００Ｖ，２ｈ，４℃)を行い、回収した溶出液からエタノール沈殿により３３.３ｋｂＤＮＡを得た。１.９ｋｂフラグメントは、ＱＩＡＧＥＮ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ(QIAGEN社製)を用いて通常の方法で溶出精製した。得られた各精製ＤＮＡフラグメントの純度をアガロース電気泳動で確認した。 The 33.3 kb fragment was eluted by electrophoresis (100 V, 2 h, 4 ° C.) from the cut gel piece using a GeBAflex-tube Dialysis Kit (MW: 3500, manufactured by WACO), and ethanol was used from the recovered eluate. 33.3 kb DNA was obtained by precipitation. The 1.9 kb fragment was eluted and purified by a usual method using QIAGEN Gel Extraction Kit (manufactured by QIAGEN). The purity of each purified DNA fragment obtained was confirmed by agarose gel electrophoresis.
　Ｔ７ゲノムのＴａｉｌ　ｆｉｂｅｒ　ｐｒｏｔｅｉｎ(ｇｐ１７)の３´末端に、ＧＳスペーサー配列(GGGGSGGGGSGGGGS)を介してｓｕｐｅｒｆｏｌｄｅｒＧＦＰを連結した融合タンパクＴａｉｌ　ｆｉｂｅｒ－ｓｆＧＦＰ(ｇｐ１７－ｓｆＧＦＰ)の遺伝子を設計した。Ｇｐ１７配列内のＳｆｉＩサイトからｇｐ１７下流Ｔ７ゲノム上のＰｍｌＩサイトまでの約２.９ｋｂフラグメント(中ほどにＧＳスペーサーおよびｓｆＧＦＰ遺伝子を含む)を、以下のオーバーラップ伸長ＰＣＲを行うことにより調製した。この調製方法を図３に示す。 We designed a gene for the fusion protein Tail fiber-sfGFP (gp17-sfGFP) in which a superfolderGFP was linked to the 3'end of the T7 genome's Tail fiber protein (gp17) via a GS spacer sequence (GGGGSGGGGSGGGGS). Approximately 2.9 kb fragments (including the GS spacer and sfGFP gene in the middle) from the SfiI site in the Gp17 sequence to the PmlI site on the gp17 downstream T7 genome were prepared by performing the following overlap extension PCR. This preparation method is shown in FIG.
　２.９ｋｂを約１ｋｂずつの３本のフラグメント(Ａ、Ｂ、Ｃ)に分けて設計し、それぞれ５´および３´末端に３０ｂｐのオーバーラップ配列を配置して、Ａ、Ｂ、Ｃの３本のＤＮＡフラグメントを化学合成によって調製した。フラグメントＡとＢのオーバーラップ伸長ＰＣＲ産物Ａ＋Ｂ(５´Ｐｒｉｍｅｒ　GTAGGTCAGGCCGTTGTGG(配列番号１))、(３´Ｐｒｉｍｅｒ　TAATCCTATCAGTGCTCCCTTCC(配列番号２))と、フラグメントＣを用いてさらにオーバーラップ伸長ＰＣＲ(５´Ｐｒｉｍｅｒ　GTAGGTCAGGCCGTTGTGG(配列番号１))、(３´Ｐｒｉｍｅｒ　GTTCTCTTCACGTGTCCTTGGGTACAG(配列番号３))を行い、目的の２.９ｋｂ産物を得た。各ＰＣＲ産物はアガロースゲル電気泳動を行って該当するバンドを切り出し、Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔを用いて溶出精製した。精製した２.９ｋｂフラグメントの５´および３´末端を制限酵素ＳｆｉＩおよびＰｍｌＩにて消化し、再度アガロースゲル電気泳動を行って(結果を図４に示す。)、Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔを用いて溶出精製した。 2.9 kb was designed by dividing it into three fragments (A, B, C) of about 1 kb each, and 30 bp overlapping sequences were arranged at the 5'and 3'ends, respectively, to form 3 of A, B, and C. The DNA fragments of the book were prepared by chemical synthesis. Overlapping extension PCR products A + B (5'Primer GTAGGTCAGGCCGTTGTGG (SEQ ID NO: 1)) and (3'Primer TAATCCTATCAGTGCTCCCTTCC (SEQ ID NO: 2)) of fragments A and B, and further overlapping extension PCR (5'Primer) using fragment C. GTAGGTCAGGCCGTTGTGG (SEQ ID NO: 1)) and (3'Primer GTTCTCTTCACGTGTCCTTGGGTACAG (SEQ ID NO: 3)) were performed to obtain the desired 2.9 kb product. Each PCR product was subjected to agarose gel electrophoresis to cut out the corresponding band, and was eluted and purified using Gel Extension Kit. The 5'and 3'ends of the purified 2.9 kb fragment were digested with restriction enzymes SfiI and PmlI, subjected to agarose gel electrophoresis again (results are shown in FIG. 4), and eluted and purified using Gel Extension Kit. did.
　実施例１で精製したＴ７ゲノムＤＮＡのＳｆｉＩおよびＰｍｌＩ切断産物である３３.３ｋｂ、１.９ｋｂの２本のフラグメントと、オーバーラップ伸長ＰＣＲ産物である２・９ｋｂフラグメントの、合計３本のフラグメントを、Ｔ４　ＤＮＡ　ｌｉｇａｓｅ(Ligation High Ver.2,TOYOBO社製)を用いて ライゲーション反応により連結した。ライゲーション反応産物の概略を図５に示す。ｉｎ　ｖｉｔｒｏ　Ｔ７　Ｐａｃｋａｇｉｎｇ　Ｋｉｔ(Merk Millipore社製)を用いて、ライゲーション反応産物とＴ７ファージ抽出物とのパッケージング反応を行い(室温２時間)、対数増殖期に達した大腸菌 ＢＬ２１(Merk Millipore社製)に感染させた。 Two fragments of 33.3 kb and 1.9 kb, which are SfiI and PmlI cleavage products of the T7 genomic DNA purified in Example 1, and a 2.9 kb fragment, which is an overlap extension PCR product, are used for a total of three fragments. , T4 DNA ligase (Ligation High Ver.2, manufactured by TOYOBO) was used for ligation reaction. The outline of the ligation reaction product is shown in FIG. Escherichia coli BL21 (manufactured by Merck Millipore) that has reached the logarithmic growth phase after packaging reaction between the ligation reaction product and T7 phage extract using in vitro T7 Packaging Kit (manufactured by Merck Millipore). Infected.
　感染させたＢＬ２１をＴＯＰ　ａｇａｒｏｓｅと混和してＬＢプレートに蒔き、出現したプラークが蛍光を発することを、実体顕微鏡による蛍光観察で、肉眼で観察して確認した。この実体顕微鏡写真を図６に示す。採取したプラークをファージ抽出緩衝液(２０ｍＭ　Ｔｒｉｓ－ＨＣｌ，１００ｍＭ　ＮａＣｌ，６ｍＭ　ＭｇＳＯ４，ｐＨ８.０)とインキュベート(４℃、３ｈ)し、少量のクロロホルムと転倒混和後、遠心(３,０００×ｇ，５分)して上清を採取し、ファージ抽出液を得た。抽出したファージ液の蛍光を実体顕微鏡下でさらに確認後、抽出したファージＤＮＡを鋳型にしてＰＣＲを行い(５‘Ｐｒｉｍｅｒ　TCAGATAACAACAATGACTGTACCTTCCAC(配列番号４))、(３‘Ｐｒｉｍｅｒ　TCTTCACGTGTCCTTGGGTACAGAGC(配列番号５))、ｇｐ１７－ｓｆＧＦＰが予想する分子量および単一バンドであること確認し、ＧＳスペーサーおよびｓｆＧＦＰ配列がＴ７ゲノムＤＮＡｇｐ１７の３´末端に挿入されていることを確認した。 The infected BL21 was mixed with TOP agarose and sown on an LB plate, and it was confirmed by visual observation with a stereomicroscope that the plaques that appeared fluoresce. This stereomicrograph is shown in FIG. The collected plaque is incubated with a phage extraction buffer (20 mM Tris-HCl, 100 mM NaCl, 6 mM sulfonyl 4 , pH 8.0) (4 ° C., 3 hours), mixed with a small amount of chloroform by inversion, and then centrifuged (3,000 × g). , 5 minutes), and the supernatant was collected to obtain a phage extract. After further confirming the fluorescence of the extracted phage solution under a stereomicroscope, PCR was performed using the extracted phage DNA as a template (5'Primer TCAGATAACAACAATGACTGTACCTTCCAC (SEQ ID NO: 4)), (3'Primer TCTTCACGTGTCCTTGGGTACAGAGC (SEQ ID NO: 5)), It was confirmed that gp17-sfGFP had the expected molecular weight and single band, and that the GS spacer and sfGFP sequence were inserted at the 3'end of T7 genomic DNA gp17.
　次に、得られたファージのゲノムＤＮＡの塩基配列解析を行い、Ｔ７ｇｐ１７－ｓｆＧＦＰファージが生成されたことを塩基配列レベルで確認した。このＴ７ｇｐ１７－ｓｆＧＦＰファージを第１世代とし、さらに大腸菌ＢＬ２１に感染させて液体培地(Ｌ－Ｂｒｏｔｈ)中で増殖させて、溶菌を確認したのち遠心(８,０００×ｇ，１０分)し、上清を採取することによりＴ７ｇｐ１７－ｓｆＧＦＰファージを得た(第２世代)。得られたＴ７ｇｐ１７－ｓｆＧＦＰファージ液の蛍光を実体蛍光顕微鏡下で確認し、上記第１世代同様、これを鋳型にしてＰＣＲを行い、ｇｐ１７－ｓｆＧＦに該当する単一バンドを確認後、さらにゲノムＤＮＡの塩基配列解析を行って、世代を経たファージ増殖後もＴ７ｇｐ１７－ｓｆＧＦＰが、ゲノム上の設計通りの位置に継代されていることを確認した。 Next, the nucleotide sequence analysis of the obtained phage genomic DNA was performed, and it was confirmed at the nucleotide sequence level that T7gp17-sfGFP phage was generated. This T7gp17-sfGFP phage was used as the first generation, further infected with Escherichia coli BL21, grown in a liquid medium (L-Bros), lysed, and then centrifuged (8,000 × g, 10 minutes) on the top. T7gp17-sfGFP phage was obtained by collecting Qing (second generation). The fluorescence of the obtained T7gp17-sfGFP phage solution was confirmed under a stereoscopic fluorescence microscope, and PCR was performed using this as a template as in the first generation above, and after confirming a single band corresponding to gp17-sfGF, further genomic DNA. It was confirmed that T7gp17-sfGFP was passaged to the designed position on the genome even after the generational phage proliferation.
　上記のように、作成したファージにｓｆＧＦＰの蛍光が認められることから、ｓｆＧＦＰは正しくフォールディングされてファージに組み込まれていることが予想された。また、上記のシークエンス解析によって、ｓｆＧＦＰはｇｐ１７にインフレームで組み込まれていることが判明した。そこで、タンパク質レベルで設計どおりに翻訳されているかどうかを以下のように調べた。作成したｇｐ１７－ｓｆＧＦＰファージをＳＤＳ－ＰＡＧＥおよびウエスタンブロットにより抗ＧＦＰ抗体で検出し、検出されるバンドの分子量が、ｇｐ１７タンパク質＋ＧＳスペーサー＋ｓｆＧＦＰの総和分子量の約９０ｋＤａ付近に認められるかどうかを調べた。 As described above, since fluorescence of sfGFP was observed in the prepared phage, it was expected that sfGFP was correctly folded and incorporated into the phage. In addition, the above sequence analysis revealed that sfGFP was incorporated into gp17 in-frame. Therefore, we investigated whether the translation was done as designed at the protein level as follows. The prepared gp17-sfGFP phage was detected by SDS-PAGE and Western blotting with an anti-GFP antibody, and it was examined whether the molecular weight of the detected band was observed near about 90 kDa of the total molecular weight of gp17 protein + GS spacer + sfGFP.
　コントロールサンプルとして、ｇｐ１７－ＧＳスペーサー－ｓｆＧＦＰタンパク質のみを大腸菌で作らせ、蛍光を発することを確認したのち、上記Ｔ７ｇｐ１７－ｓｆＧＦＰファージと同様に、ＳＤＳ－ＰＡＧＥおよびウエスタンブロットして抗ＧＦＰ抗体で検出した。結果は、大腸菌で作成したｇｐ１７－ＧＳスペーサー－ｓｆＧＦＰタンパク質と、Ｔ７ｇｐ１７－ｓｆＧＦＰファージとで同じ約９０ｋＤａ付近に抗ＧＦＰ抗体で認識されるバンドが検出された(図７)。このことから、上記ＧＦＰの蛍光を発するＴ７ｇｐ１７－ｓｆＧＦＰファージは、塩基配列レベルおよびタンパク質レベル双方において、設計どおりに構築されていること、そして実際に強い蛍光を認めることから、ｓｆＧＦＰは正しくフォールディングされてＴ７ファージの尾部繊維タンパク質に組み込まれていることが確認された。 As a control sample, only the gp17-GS spacer-sfGFP protein was produced in Escherichia coli, and after confirming that it fluoresces, it was detected by SDS-PAGE and Western blotting with an anti-GFP antibody in the same manner as the above T7gp17-sfGFP phage. .. As a result, a band recognized by the anti-GFP antibody was detected in the same vicinity of about 90 kDa between the gp17-GS spacer-sfGFP protein prepared in Escherichia coli and the T7gp17-sfGFP phage (Fig. 7). From this, the T7gp17-sfGFP phage that fluoresces the above GFP is constructed as designed at both the base sequence level and the protein level, and actually shows strong fluorescence, so that the sfGFP is folded correctly. It was confirmed that it was integrated into the tail fiber protein of T7 phage.
[組換えファージのファージライブラリーの作製]
　実施例３で作製したＴ７ｇｐ１７－ｓｆＧＦＰファージを、ＰＥＧ沈殿により濃縮した後２％ＳＤＳ処理によりファージゲノムＤＮＡを抽出し、１６，５００×ｇで遠心した上清から、ＱＩＡＧＥＮ　Ｇｅｎｏｍｉｃ－ｔｉｐ　２０/Ｇ(ＱＩＡＧＥＮ社)を用いてＴ７ｇｐ１７－ｓｆＧＦＰファージゲノムＤＮＡ(３８．１ｋｂ)を精製した。得られたファージゲノムＤＮＡをエタノール沈殿後、ＥｃｏＲＩおよびＨｉｎｄＩＩＩで消化し、２１．５ｋｂｐおよび１６．６ｋｂｐの断片を得、０．８％アガロースゲル電気泳動を行って、ＱＩＡＧＥＮ　ＧＥＬ　ＥＸＴＲＡＣＴＩＯＮ　ＫＩＴを用いて精製した（図８）。 [Preparation of phage library of recombinant phage]
The T7gp17-sfGFP phage prepared in Example 3 was concentrated by PEG precipitation, then the phage genomic DNA was extracted by 2% SDS treatment, and the supernatant was centrifuged at 16,500 × g. T7gp17-sfGFP phage genomic DNA (38.1 kb) was purified using QIAGEN). The obtained phage genomic DNA was precipitated with ethanol and then digested with EcoRI and HindIII to obtain 21.5 kbp and 16.6 kbp fragments, subjected to 0.8% agarose gel electrophoresis and purified using QIAGEN GEL EXTRATION KIT. (Fig. 8).
　ファージライブラリーのランダムペプチド(１２アミノ酸残基)に該当する配列とスペーサー配列および６×Ｈｉｓタグ配列、ＦＬＡＧタグ配列を含む１５０ｂｐのＤＮＡ配列(GGCGGCGGTGGTCATCACCATCACCATCATGGCGGTGGGTCGGGTGGCGGGTCTGGTGGCGGGTCAGGCGGTGGCNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKGGTGGCGGTGGCGGGGACTACAAAGACGATGACGACAAG(配列番号６))を合成し、これをテンプレートとしてＥｃｏＲＩ配列を含む(５′プライマー　TATGAATTCGGGCGGCGGTGGTCATCACCATCAC((配列番号７))およびＨｉｎｄＩＩＩ配列を含む(３′プライマー　GCGAAGCTTACTTGTCGTCATCGTCTTTGTAGTC(配列番号８))を用いてＰＣＲ反応を行い、ＰＣＲ産物として、ファージゲノムＤＮＡに挿入するインサートＤＮＡを得た。このＰＣＲ産物の５′および３′末端をＥｃｏＲＩおよびＨｉｎｄＩＩＩで消化した後、２％アガロース電気泳動およびＱＩＡＧＥＮ　ＧＥＬ　ＥＸＴＲＡＣＴＩＯＮ　ＫＩＴにより精製した。得られたインサートＤＮＡと、２１．５ｋｂｐおよび１６．６ｋｂｐのファージゲノムＤＮＡをＴ４ライゲースによりライゲーション反応を行い、ライゲーション産物をＴ７　Ｐａｃｋａｇｉｎｇ　Ｅｘｔｒａｃｔｓ（メルクミリポア社）と混合して、Ｉｎ　ｖｉｔｒｏ　ｐａｃｋａｇｉｎｇ反応を行った。Ｐａｃｋａｇｉｎｇ産物を大腸菌ＢＬ２１に感染させて、ＬＢプレート(１５０ｍｍ)に蒔いて増幅させ(３７℃、２．５時間)、増幅したファージを、１００ｍＭ　ＮａＣｌおよび６ｍＭ　ＭｇＳＯ４を含む緩衝液でプラークから回収し、遠心した上清を回収して、Ｔ７ｇｐ１７－ｓｆＧＦＰランダムペプチドライブラリー(Ｔ７－ｓｆＧＦＰ×１２)を得た。 150bp DNA sequence including sequence corresponding to random peptide (12 amino acid residues) of phage library, 6 × His tag sequence, FLAG tag sequence (GGCGGCGGTGGTCATCACCATCACCATCATGGCGGTGGGTCGGGTGGCGGGTCTGGTGGCGGGTCAGGCGGTGGCNNKNNKNNKGGCGGGTCAGGCGGTGGCNNKNNKNNKGGT PCR reaction was performed using the EcoRI sequence (5'primer TATGAATTCGGGCGGCGGTGGTCATCACCATCAC ((SEQ ID NO: 7))) and the HindIII sequence (3'primer GCGAAGCTTACTTGTCGTCATCGTCTTTGTAGTC (SEQ ID NO: 8)), and inserted into the phage genomic DNA as a PCR product. The 5'and 3'ends of this PCR product were digested with EcoRI and HindIII and then purified by 2% agarose electrophoresis and QIAGEN GEL EXTRATION KIT. The obtained insert DNA and 21.5 kbp And 16.6 kbp phage genomic DNA was subjected to a ligation reaction with T4 ligation, the ligation product was mixed with T7 Packing Exects (Merck Millipore), and an In vitro packing reaction was carried out. The packing product was infected with Escherichia coli BL21. , Sowed on an LB plate (150 mm) and amplified (37 ° C., 2.5 hours), the amplified phage was recovered from the plaque with a buffer containing 100 mM NaCl and 6 mM Then 4 and the centrifuged supernatant was recovered. , T7gp17-sfGFP random peptide library (T7-sfGFP × 12) was obtained.
[ランダムペプチドファージライブラリーを用いたｉｎ　ｖｉｔｒｏスクリーニング]
　動物細胞発現用プラスミドベクターｐＣＡＧＥＮ(Ａｄｄｇｅｎｅ社)のマルチクローニングサイトに、マウスＬＤＬ受容体(ｍＬＤＬＲ)の細胞外領域(ｅｘｔ)(ｍＬＤＬＲｅｘｔ)とその３’末端にｍｙｃタグ配列を連結させた断片(ｍＬＤＬＲｅｘｔ－ｍｙｃ)を挿入した動物細胞発現ベクタ―を作成した。これをＨＥＫ２９３－Ｔ細胞にトランスフェクションし、４８時間後に培養上清にｍＬＤＬＲｅｘｔ－ｍｙｃが分泌されていることを、抗ｍｙｃ抗体(マウスＩｇＧ)を用いてウエスタンブロットにて確認した(図９)。この培養上清から、抗ｍｙｃ抗体(マウスＩｇＧ)および抗マウスＩｇＧ－磁気ビーズ(Ｄｙｎａｂｅａｄｓ　Ｍ－２８０　Ｓｈｅｅｐ　ａｎｔｉ－Ｍｏｕｓｅ　ＩｇＧ、ベリタス社)を用いて、免疫沈降法によりｍＬＤＬＲｅｘｔ－ｍｙｃ－磁気ビーズを作成した。これをリガンドとして、上記ランダムペプチドライブラリー(Ｔ７－ｓｆＧＦＰ×１２)と４℃、１時間インキュベートし、緩衝液で洗浄したのちに、ｍＬＤＬＲｅｘｔ－ｍｙｃ－磁気ビーズに結合したファージプールを得、１％ＳＤＳで溶出して、ＬＤＬ受容体細胞外領域(ＬＤＬＲｅｘｔ)に結合性を示すファージの１次プールとした(ラウンド１)。
　これを大腸菌ＢＬ２１に感染させて増幅し、ラウンド１のインプットと同じ量のファージを次のラウンド２のスクリーニングのインプットとして用い、上記同様の操作を続けて、合計３回のバイオパニングを実施した。ラウンドを進めるにつれて回収されるファージの「アウトプット／インプット」の比が上昇し、最終的には、ＬＤＬＲｅｘｔ結合性を示すファージの占める割合がラウンド１の１０００倍以上に上昇した(図１０)。 [In vitro screening using random peptide phage library]
A fragment (mLDLRext) in which the extracellular region (ext) (mLDLRext) of the mouse LDL receptor (mLDLR) and the myc tag sequence are linked to the 3'end of the multicloning site of the plasmid vector pCAGEN (Addgene) for animal cell expression An animal cell expression vector into which -myc) was inserted was prepared. This was transfected into HEK293-T cells, and it was confirmed by Western blotting using an anti-myc antibody (mouse IgG) that mLDLRext-myc was secreted into the culture supernatant 48 hours later (Fig. 9). From this culture supernatant, mLDLRext-myc-magnetic beads were prepared by immunoprecipitation using anti-myc antibody (mouse IgG) and anti-mouse IgG-magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG, Veritas). did. Using this as a ligand, it was incubated with the above random peptide library (T7-sfGFP × 12) at 4 ° C. for 1 hour, washed with a buffer solution, and then a phage pool bound to mLDLRext-myc-magnetic beads was obtained, and 1%. It was eluted with SDS to form a primary pool of phage exhibiting binding to the extracellular region of the LDL receptor (LDLLext) (Round 1).
This was infected with Escherichia coli BL21 and amplified, and the same amount of phage as the input of Round 1 was used as the input for the screening of the next Round 2, and the same operation as described above was continued, and biopanning was performed a total of 3 times. As the round progressed, the "output / input" ratio of the recovered phage increased, and finally, the proportion of phage showing LDLRext binding increased to more than 1000 times that of Round 1 (Fig. 10).
　得られたラウンド３のファージをＰＥＧ沈殿により濃縮し、ＨＥＫ２９３－Ｔ細胞培養液(ＤＭＥＭ、１０％ＦＣＳ)に懸濁し、マウスＬＤＬ受容体(細胞内Ｃ末端にｍＣｈｅｒｒｙを連結)を発現させたＨＥＫ２９３－Ｔ細胞の培養上清に添加した。室温で１０－１５分のインキュベーション後、蛍光顕微鏡で観察すると、ファージはｍＬＤＬＲ－ｍＣｈｅｒｒｙ発現細胞に特異的に結合することが確認された(図１１)。図１１中、ａ、ＧＦＰシグナル(緑)；ｂ、ｍＣｈｅｒｒｙシグナル(赤)；ｃ、ａとｂの重ね合わせ。 The obtained round 3 phage was concentrated by PEG precipitation, suspended in HEK293-T cell culture medium (DMEM, 10% FCS), and expressed in mouse LDL receptor (mCherry linked to intracellular C-terminal) HEK293. -Added to the culture supernatant of T cells. After incubation for 10-15 minutes at room temperature, observation with a fluorescence microscope confirmed that the phage specifically bound to mLDLR-mCherry-expressing cells (Fig. 11). In FIG. 11, a, GFP signal (green); b, mCherry signal (red); c, a and b are superposed.
　以上、図１０および図１１の結果は、本発明により創出した新規蛍光ファージＴ７ｇｐ１７－ｓｆＧＦＰは、（１）これを用いてランダムペプチド蛍光ファージライブラリーの作製が可能であること、（２）目的とする機能ペプチド配列を有する蛍光ファージが標的へ結合する様子を、ライブイメージングすることが可能であることを示す。
　このように、本発明のファージの放つ蛍光を検知することにより、受容体などの標的に結合性を示す機能ペプチド配列を持つファージをリアルタイムで追尾しながら、バイオパニングを迅速に遂行することができ、目的とする機能ペプチドの創出を、従来のファージを用いたスクリーニング・バイオパニングに比べて、極めて高速に創出することが可能となることを示すものである。 As described above, the results of FIGS. 10 and 11 show that the novel fluorescent phage T7gp17-sfGFP created by the present invention can (1) be used to prepare a random peptide fluorescent phage library, and (2) purpose. It is shown that it is possible to perform live imaging of how fluorescent phage having a functional peptide sequence binds to a target.
In this way, by detecting the fluorescence emitted by the phage of the present invention, biopanning can be rapidly performed while tracking the phage having a functional peptide sequence exhibiting binding to a target such as a receptor in real time. This shows that the production of the target functional peptide can be performed at an extremely high speed as compared with the conventional screening / biopanning using phages.
[ランダムペプチドファージライブラリーを用いたｉｎ　ｖｉｖｏスクリーニング]
　Ｔ７ｇｐ１７－ｓｆＧＦＰランダムペプチドライブラリー(Ｔ７－ｓｆＧＦＰ×１２)を、麻酔したマウス鼻粘膜に滴下し、一定時間後に脳を摘出し、ホモジナイズして大腸菌ＢＬ２１に感染させ、ＬＢプレート(１５０ｍｍ)にまいて増幅(３７℃、２．５時間)後、プラークからファージを回収した。このファージを用いて次のラウンドへ進み、ラウンド１と等量のファージをマウス鼻粘膜に滴下し、上記同様の操作を４回繰り返した。ラウンドを進めるにつれて回収されるファージの「アウトプット／インプット」の比が約９００倍上昇した(図１２)。 [In vivo screening using random peptide phage library]
The T7gp17-sfGFP random peptide library (T7-sfGFP × 12) was dropped onto the nasal mucosa of anesthetized mice, and after a certain period of time, the brain was removed, homogenized and infected with Escherichia coli BL21, and spread on an LB plate (150 mm). After amplification (37 ° C., 2.5 hours), phage was recovered from the plaque. Using this phage, the process proceeded to the next round, and the same amount of phage as in Round 1 was dropped onto the nasal mucosa of the mouse, and the same operation as described above was repeated 4 times. As the round progressed, the “output / input” ratio of the recovered phage increased about 900-fold (Fig. 12).
　ラウンド４で得られたファージから任意に約１００クローンを選択し、ファージＤＮＡのランダムペプチド部分に該当する塩基配列を、ＢｉｇＤｙｅＴＭＴｅｒｍｉｎａｔｏｒ　ｖ３．１　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ(ThermoFisher社)を用いて解析した。得られた塩基配列のうち、出現頻度の極めて高い塩基配列を持つファージクローン群から１つを選んで(クローンＡ)、ペプチド該当部分の１２アミノ酸残基のペプチドを化学合成し、Ｎ末端にＴＡＭＲＡ蛍光標識を行った。 Approximately 100 clones were arbitrarily selected from the phages obtained in Round 4, and the nucleotide sequence corresponding to the random peptide portion of the phage DNA was analyzed using a BigDye TM Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher). From the obtained nucleotide sequences, one is selected from a group of phage clones having an extremely frequent nucleotide sequence (clone A), a peptide having 12 amino acid residues of the peptide-corresponding portion is chemically synthesized, and TAMRA at the N-terminal. Fluorescent labeling was performed.
　上記の末端ＴＡＭＲＡ化したペプチドをＰＢＳに溶解し麻酔下のマウスの鼻粘膜に滴下(１ｍＭ、５μＬ)、１～２時間後に４％パラホルムアルデヒド・リン酸緩衝液を用いて灌流固定を行った。断頭後に頭皮を除去して落射型蛍光実体顕微鏡(OLYMPUS SZX16)下、低倍率でＴＡＭＲＡシグナルの観察を頭蓋骨の上から行った。ＴＡＭＲＡシグナルは嗅球付近に強いシグナルを骨の外から検出できた(図１３)。 The above terminal TAMRA-ized peptide was dissolved in PBS and dropped onto the nasal mucosa of anesthetized mice (1 mM, 5 μL), and after 1 to 2 hours, perfusion fixation was performed using 4% paraformaldehyde / phosphate buffer. After decapitation, the scalp was removed and the TAMRA signal was observed from above the skull under an epi-illumination stereomicroscope (OLYMPUS SZX16) at low magnification. As for the TAMRA signal, a strong signal near the olfactory bulb could be detected from outside the bone (Fig. 13).
　上記観察後、０．５Ｍ　ＥＤＴＡ溶液(ｐＨ８．０)を用いて脱カルシウム処理(４℃、ｏｖｅｒｎｉｇｈｔ×７ｄａｙｓ)を行い，３０％スクロース・リン酸緩衝液に置換後、頭骨ごと凍結切片(５０μｍ　ｔｈｉｃｋ、矢状断)を作製して落射型蛍光顕微鏡(OLYMPUS
BX53)を用いてＴＡＭＲＡシグナルの観察を行った。結果を図１４に示す。鼻粘膜から嗅上皮および呼吸上皮の神経細胞内(図１４(ａ)(ｂ))に侵入した機能ペプチドの強いシグナルを検出した。
　以上、実施例５および６の結果は、本発明にて創出した新規蛍光ファージＴ７ｇｐ１７－ｓｆＧＦＰ、およびこれを用いて作成したランダムペプチド蛍光ファージライブラリーがｉｎ　ｖｉｔｒｏだけでなくｉｎ　ｖｉｖｏにおいてもスクリーニングやバイオパニングに応用が可能であることを示しており、生体反応を利用した機能ペプチド配列スクリーニングを、ｉｎ　ｖｉｖｏでファージの蛍光をリアルタイムに追尾しながら極めて迅速に遂行することが可能になることを示すものである。 After the above observation, decalcification treatment (4 ° C., overnight × 7 days) was performed using 0.5 M EDTA solution (pH 8.0), replaced with 30% sucrose / phosphate buffer, and then frozen sections (50 μm microscope) together with the occipital bone. , Sagittal section) and epi-illumination fluorescence microscope (OLYMPUS)
The TAMRA signal was observed using BX53). The results are shown in FIG. Strong signals of functional peptides that invaded the olfactory epithelium and respiratory epithelium neurons (FIGS. 14 (a) and 14 (b)) from the nasal mucosa were detected.
As described above, the results of Examples 5 and 6 show that the novel fluorescent phage T7gp17-sfGFP created in the present invention and the random peptide fluorescent phage library prepared using the same are screened and bio-screened not only in vitro but also in vivo. It shows that it can be applied to panning, and shows that functional peptide sequence screening using biological reactions can be performed extremely quickly while tracking phage fluorescence in vivo in real time. Is.
　本発明の組換えバクテリオファージＴ７は、ファージ分子あたりの発現する標識タンパク質である蛍光タンパク質の数が一定であり、数も多くて蛍光強度が高いので、正確な一定の蛍光強度によるスクリーニングを実現することができる。
　また、本発明の組換えファージをファージディスプレイに使用する場合に、ライブラリー部分のランダムペプチドを発現するキャプシド部分とは異なる構造部分に蛍光タンパク質等が発現することから、ターゲットに対する結合の立体障害を生じることなく、ファージディスプレイ法で重要な要素であるターゲットとの結合の見逃しが起きないという優れた効果を有する。 The recombinant bacteriophage T7 of the present invention has a constant number of fluorescent proteins, which are labeling proteins expressed per phage molecule, and has a large number and high fluorescence intensity, so that screening with an accurate constant fluorescence intensity is realized. be able to.
In addition, when the recombinant phage of the present invention is used for phage display, a fluorescent protein or the like is expressed in a structural part different from the capsid part expressing the random peptide in the library part, so that the steric disorder of binding to the target is caused. It has an excellent effect that the binding to the target, which is an important element in the phage display method, is not overlooked without occurring.
　本発明の蛍光ファージは、自身の放つ蛍光によってリアルタイム検出ができるので、従来法のＥＬＩＳＡなどを用いた間接的で時間と手間のかかるファージ検出法を省略できるのみならず、先行研究のように蛍光タンパク質をヘルパープラスミドを用いた大腸菌による過剰発現でファージに付加しないので、スクリーニングにおけるパニングの過程で、選択したファージを複製・増幅するラウンドごとに、未結合蛍光タンパク質と目的の蛍光を発するファージとを分離する手間もかからない。選択増幅したすべてのファージに蛍光タンパク質が間違いなく結合しているかを、ラウンドのたびに検証する必要がなく、一定強度のファージの極めて迅速かつ正確な選択増幅によるスクリーニングを実現できる。 Since the fluorescent phage of the present invention can be detected in real time by its own fluorescence, not only can the indirect, time-consuming and time-consuming phage detection method using the conventional method such as ELISA be omitted, but also fluorescence as in the previous research. Since the protein is not added to the phage by overexpression by E. coli using a helper plasmid, the unbound fluorescent protein and the phage that emits the desired fluorescence are selected for each round of replication and amplification of the selected phage during the panning process in the screening. There is no need to separate them. It is not necessary to verify that the fluorescent protein is definitely bound to all the selectively amplified phages every round, and it is possible to realize a very rapid and accurate selective amplification screening of phages of constant intensity.
　さらに、非特許文献２では、蛍光タンパク質とＴ７キャプシドタンパク質の融合タンパク質をヘルパープラスミドを用いて大腸菌による過剰発現でファージに付加しているが、この方法では、野生型のＴ７キャプシドタンパク質をさらに大過剰に同時に発現させることがファージ生成に必須であり、このことにより、実際は全ファージの一部にしか蛍光タンパク質が入らない(６％～１６％)。すなわち、この方法を用いると、スクリーニングにより選択して得た陽性ファージ群を次のスクリーニングのラウンドのために複製・増幅しても、全ファージの一部(６％～１６％)にしか蛍光が入らず、残りの大部分(８４％～９４％)は選択済みの陽性ファージ集団であるにも関わらず、蛍光の無いファージである。選択したファージは、元々ランダムペプチドライブラリーから選択したヘテロな配列のファージ集団なので、この蛍光が入らなかった８４％～９４％のファージ群の中に、最も目標とする配列を持つファージが含まれてしまう可能性は極めて高く、蛍光タンパク質を大腸菌による過剰発現で付加する方法では、目標とするペプチド配列を持つファージを見逃す可能性が高い。 Further, in Non-Patent Document 2, a fusion protein of fluorescent protein and T7 capsid protein is added to phage by overexpression by Escherichia coli using a helper plasmid, but in this method, wild-type T7 capsid protein is further excessively excessive. Simultaneous expression in E. coli is essential for phage production, which actually results in only a portion of all phages containing the fluorescent protein (6% -16%). That is, using this method, even if the positive phage group selected by screening is replicated and amplified for the next screening round, only a part (6% to 16%) of all phages are fluorescent. Most of the remaining (84% -94%) are non-fluorescent phages, even though they are selected positive phage populations. Since the selected phage is a heterogeneous sequence of phage population originally selected from the random peptide library, the 84% to 94% of phage groups that did not receive this fluorescence include the phage having the most targeted sequence. In the method of adding the fluorescent protein by overexpression by E. coli, there is a high possibility that the phage having the target peptide sequence will be missed.
　しかも、６％～１６％の蛍光が入ったファージ群でも、有する蛍光タンパク質は１ファージ当たり１個から最大３個相当なので、前述のように蛍光そのものが弱く強度が一定していないので、スクリーニングには適さない。本発明では、スクリーニングで選択したファージは、選択と増幅のラウンドを重ねてもすべてのファージに蛍光が入った状態で増幅され、陽性ファージの取りこぼしがない。すべてが一定数１８個の蛍光タンパク質を有し、極めて高度の蛍光強度を有することからも、ｉｎ　ｖｉｔｒｏのみならずｉｎ　ｖｉｖｏでのスクリーニングに極めて有用なものである。
  Moreover, even in the phage group containing 6% to 16% of fluorescence, the number of fluorescent proteins contained in each phage is equivalent to 1 to 3 at maximum, and as described above, the fluorescence itself is weak and the intensity is not constant, so that it is suitable for screening. Is not suitable. In the present invention, the phages selected by screening are amplified with fluorescence in all the phages even if the selection and amplification rounds are repeated, and positive phages are not missed. Since all of them have a certain number of 18 fluorescent proteins and have extremely high fluorescence intensity, they are extremely useful for screening not only in vitro but also in vivo.

Claims (10)

  1. 　組換えバクテリオファージＴ７であって、バクテリオファージＴ７のゲノムのｇｐ１７遺伝子領域に、標識タンパク質をコードする遺伝子が発現可能に挿入されていることを特徴とする、組換えバクテリオファージＴ７。 Recombinant bacteriophage T7, characterized in that a gene encoding a labeled protein is expressively inserted into the gp17 gene region of the genome of the recombinant bacteriophage T7.
  2. 　前記標識タンパク質が、蛍光タンパク質または発光または呈色反応を触媒する酵素である、請求項１に記載の組換えバクテリオファージＴ７。 The recombinant bacteriophage T7 according to claim 1, wherein the labeled protein is a fluorescent protein or an enzyme that catalyzes a luminescence or color reaction.
  3. 　前記蛍光タンパク質が緑色蛍光タンパク質（ＧＦＰ）であり、前記発光または呈色反応を触媒する酵素が、ルシフェラーゼ、β－グルクロニダーゼ（ＧＵＳ）、β－ガラクトシダーゼ、西洋ワサビペルオキシダーゼのいずれか一つである、請求項２に記載の組換えバクテリオファージＴ７。 Claimed that the fluorescent protein is green fluorescent protein (GFP) and the enzyme that catalyzes the luminescence or color reaction is one of luciferase, β-glucuronidase (GUS), β-galactosidase, and horseradish peroxidase. Item 2. The recombinant bacterophage T7 according to Item 2.
  4. 　前記標識タンパク質の遺伝子が、ｇｐ１７遺伝子コード領域末端近傍に挿入されている、請求項１に記載の組換えバクテリオファージＴ７。 The recombinant bacteriophage T7 according to claim 1, wherein the gene of the labeled protein is inserted near the end of the gp17 gene coding region.
  5. 　前記ｇｐ１７遺伝子コード領域末端近傍が、３´末端領域である、請求項４に記載の組換えバクテリオファージＴ７。 The recombinant bacteriophage T7 according to claim 4, wherein the vicinity of the terminal of the gp17 gene coding region is the 3'terminal region.
  6. 　組換えバクテリオファージＴ７が、宿主細胞に感染後の細胞質におけるファージ複製の間に、前記蛍光タンパク質の遺伝子または発光または呈色反応を触媒する酵素の遺伝子を発現して、活性を有する蛍光タンパク質または発光または呈色反応を触媒する酵素生成物を生じる、請求項２に記載の組換えバクテリオファージＴ７。 Recombinant bacteriophage T7 expresses the gene for the fluorescent protein or the gene for an enzyme that catalyzes a luminescence or color reaction during phage replication in the cytoplasm after infection of the host cell, resulting in an active fluorescent protein or luminescence. Alternatively, the recombinant bacteriophage T7 according to claim 2, which produces an enzyme product that catalyzes a color reaction.
  7. 　前記蛍光タンパク質または発光または呈色反応を触媒する酵素が、バクテリオファージＴ７の尾部に発現する、請求項６に記載の組換えバクテリオファージＴ７。 The recombinant bacteriophage T7 according to claim 6, wherein the fluorescent protein or an enzyme that catalyzes a luminescence or color reaction is expressed in the tail of the bacteriophage T7.
  8. 　組換えバクテリオファージＴ７であって、バクテリオファージＴ７のゲノムのｇｐ１７遺伝子領域に標識タンパク質の遺伝子が発現可能に挿入されている組換えバクテリオファージＴ７のゲノムのｇｐ１０遺伝子領域に、ランダムペプチドをコードする核酸分子を挿入した、組換えバクテリオファージＴ７のランダムペプチドファージライブラリー。 Recombinant bacteriophage T7, a nucleic acid encoding a random peptide in the gp10 gene region of the recombinant bacteriophage T7 genome, in which the gene for the labeled protein is expressively inserted into the gp17 gene region of the bacteriophage T7 genome. Random peptide phage library of recombinant bacteriophage T7 with molecules inserted.
  9. 　請求項８に記載の組換えバクテリオファージＴ７のランダムペプチドファージライブラリーを使用するｉｎ　ｖｉｔｒｏスクリーニング方法。 An in vitro screening method using the random peptide phage library of recombinant bacteriophage T7 according to claim 8.
  10. 　請求項８に記載の組換えバクテリオファージＴ７のランダムペプチドファージライブラリーを使用するｉｎ　ｖｉｖｏスクリーニング方法。
          An in vivo screening method using the random peptide phage library of recombinant bacteriophage T7 according to claim 8.
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